STRESS SIGNALING PATHWAYS LINKING ENDOTHELIAL INJURY TO GRAFT ARTERIOSCLEROSIS Abstract: The overall hypothesis of this application is that graft arteriosclerosis (GA), the major cause of late cardiac allograft failure, results from a chronic host T cell response to allogeneic graft endothelial cells (ECs) that takes the form of delayed-type hypersensitivity DTH within the vessel wall, locally generating IFN-? which is responsible for driving vascular smooth muscle cell (VSMC) proliferation and intimal hyperplasia. The clinical correlations and evidence from other experimental systems have suggested that non-immune factors, especially peri-operative stress-induced alterations in the graft, are important contributors to GA pathogenesis. It is proposed and demonstrated experimentally that signals in the graft, primarily from ECs, generated as a result of peri-operative stress can produce mediators that influence T cell activation and differentiation. However, how the peri-operative stresses such as hypoxia couple intracellular signaling pathway to alter ECs alloimmunity and GA is not understood. We have identified SENP1 is a critical mediator of peri-operative stresses. We hypothesize that SENP1 mediates the responses to non-immune peri-operative injuries of graft ECs, increasing T cell-mediated alloimmunity and GA. We propose to explore this hypothesis in the following specific aims: 1) Characterize SENP1-enhanced cytosolic NOX2 activity that mediates peri-operative stress- induced EC immunogenicity and GA progression. We will elucidate the mechanisms by which SENP1 activates NOX2-dependent ROS generation in ECs, define the role of SENP1-NOX2 axis in peri-operative stress-exacerbated GA progression in vivo using EC-specific SENP1 knockout mice and NOX2-deficient as graft donors, and determine how SENP1 couples NOX2 deSUMOylation to EC phenotypic changes. 2) Characterize SENP1-mediated deSUMOylation and disruption of mitochondrial Trx2 activity that augments peri-operative stress-induced EC immunogenicity and GA progression. We will elucidate the mechanisms by which SENP1 attenuates Trx2-dependent mitochondrial function in EC, define the role of mitochondrial SENP1-Trx2 axis in peri-operative stress-exacerbated GA progression in vivo using EC-specific Trx2- transgenic mice expressing WT, SUMO-defective KR mutant and Trx2-SUMO fusion forms as graft donors, and determine how SENP1 couples Trx2 deSUMOylation, mitochondrial dysfunction and ROS production to EC phenotypic changes that modulate T cell responses. 3) Characterize SENP1-ROS-ASK1 signaling pathway that mediates peri-operative stress-induced EC immunogenicity and GA progression. We will determine if cytosolic SENP1-NOX2 and mitochondrial SENP1-Trx2 converge on ASK1 in regulating EC function and GA progression, determine how SENP1-NOX2 and SENP1-Trx2 couple ASK1 to EC phenotypic changes that modulate T cell responses, and test if pharmacological inhibitors of SENP1, NOX2, TRX2 and SENP1 could prevent/ameliorate GA. If successful, this study will provide therapeutic strategies by modulating these two molecules in ECs to reduce GA incidence or delay GA progression.